Preweaned cancer prone ApcMin/+ (Min) mice were treated continuously with the DNA methylase inhibitor,zebularine, in their drinking water to determine the effects of the drug on normal mouse development as well as cancer prevention. Zebularine caused a tissue-specific reduction in DNA methylation at B1 short interspersed nucleotide elements in the small and large intestines of female Min mice but not in other organs examined after chronic oral treatment. No significant difference in the average weights of mice was observed during the treatment. In addition, analysis of global gene expression of colonic epithelial cells from the females indicated that only 3% to 6% of the genes were affected in their expression. We did not detect toxicity and abnormalities from the histopathologic analysis of liver and intestinal tissues. Lastly, we tested whether prevention of tumorigenesis can be achieved with chronic oral administration of zebularine in Min mice. The average number of polyps in Min females decreased from 58 to 1, whereas the average polyp number remained unaffected in Min males possibly due to differential activity of aldehyde oxidase. Taken together, our results show for the first time that long-term oral administration of zebularine causes a gender-specific abrogation of intestinal tumors while causing a tissue specific DNA demethylation. Importantly, prolonged treatment of mice with epigenetic drugs resulted in only minor developmental and histologic changes. Previous published studies have not addressed the long-term toxicity of zebularine and have mainly dealt with the anticancer properties of the drug. Therefore, it is important to explore the effects of zebularine in the entire animal following chronic administration of the methylation inhibitor. The effect of chronic DNA methylation inhibition in mice should suggest whether there is a potential for safe long-term therapy in man. The importance of this paper merited the publication of a Perspective in the same issue (Cancer Prevention Research 2008, 1, 219-222) that highlighted the results obtained in our work emphasizing that there is now strong evidence for an epigenetic component of early neoplasia, and data on the cancer preventive properties of epigenetic modulation are emerging. With preclinical proof of principle at hand, the challenge is to translate these epigenetic findings into testable clinical hypotheses. During this period we have also investigated the DNA incorporation of zebularine as the corresponding 2'-deoxy-5'-triphosphate, the hybridization properties of modified oligos and its role as a template-coding nucleobase (Nucleosides Nucleotides & Nucleic Acids 2008, 27, 131-145 and Chemistry & Biodiversity, 2008, in press). Finally, we have also succeeded in the reactivation of p16 gene silenced by DNA methylation with prodrug phosphoramidite derivatives of 2'-deoxyzebularine (J. Med. Chem. manuscript submitted).